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Identificación de proteínas que interaccionan con CypA, CypB y FKBP12 y su implicación en la toxicidad renal producida por los inmunosupresores CsA y FK506Suñé Rodríguez, Guillermo 17 December 2008 (has links)
La Ciclosporina A (CsA) es un fármaco inmunosupresor que ha supuesto una revolución en el trasplante de órganos. A pesar de sus propiedades anti-inflamatorias, su uso se ha visto limitado por los efectos tóxicos que causa en algunos pacientes. La CsA inhibe la trascripción de genes involucrados en el sistema inmune. Se cree que esta inhibición es la causante de los efectos tóxicos producidos por el fármaco. El modo de acción de CsA está mediado por la unión de sus principales receptores intracelulares, las ciclofilinas (Cyps). Las ciclofilinas son proteínas que están conservadas en todas las especies y su principal función está involucrada en el plegamiento correcto de otras proteínas. Se ha descrito que los efectos tóxicos producidos por CsA podían estar mediados directamente por sus receptores las ciclofilinas. A pesar de que se descrito que las ciclofilinas están involucradas en diferentes funciones, no se les atribuye una función específica. En nuestro laboratorio hemos estado interesados en las posibles vías que involucran a las Cyps y si éstas tienen relación con lo efectos causados por CsA. En esta tesis se ha intentado identificar proteínas que interaccionan con CypA, CypB y FKBP12 y su implicación en los efectos tóxicos producidos por CsA. Hemos identificado una serie de interacciones y hemos estudiado más a fondo dos de ellas. Estas interacciones son las ocurridas entre la ciclofilina B y la subunidad beta de la bomba sodio/potasio, la ciclofilina A y la subunidad beta de la bomba sodio/potasio y por último la ciclofilina B y la subunidad b1 de la ATP sintetasa mitocondrial. Una vez identificadas estas interacciones hemos realizado ensayos funcionales con cada una de ellas. Por un lado hemos estudiado si la actividad de la bomba sodio/potasio estaba afectada en células renales humanas tratadas con CsA y células que habían sido silenciadas para la ciclofilina B y ciclofilina A. Por otro lado hemos realizado un estudio de la actividad y expresión de los complejos de la cadena respiratoria mitocondrial en células renales tratadas con CsA y células renales que habían sido silenciadas para los genes CypA y CypB. A parte de las interacciones encontradas, hemos visto que la actividad de la bomba Na/K está disminuida por el fármaco y que en esta disminución estaría involucrada la ciclofilina B, también hemos visto que los complejos de la cadena respiratoria mitocondrial estarían afectados en células tratadas con CsA y en células interferidas tanto para CypB como para CypA. Como conclusión podríamos sugerir que las ciclofilinas estarían involucradas en los efectos nefrotóxicos que produce la CsA. Estos efectos nefrotóxicos causados por el tratamiento con CsA podrían estar mediados por vias alternativas a la clásicamente descrita, como es la inhibición de la calcineurina. Dichas vías estarían integradas por nuevos efectores tales como Na/K-ATPasa, ATP sintetasa, así como otras putativas dianas que en el futuro serán estudiadas en nuestro laboratorio. / Cyclosporine A is an immunosuppressive drug that has revolutionized organ transplantation. Even though it has anti-inflammatory property, its used has been reduced due to the renal toxic effects caused in some patients. CsA inhibits transcription of genes involved in the immune system. The CsA mode of action involves the binding of its main receptors, the cyclophilins (Cyps). Cyps are proteins conserved in all species and their main function is the correct folding of immature proteins. It has been describes that the main toxic effects caused by CsA could be mediated directly by its receptors, the cyclophilins. Although Cyps are involved in many processes, there is not a specific function for them. In our lab, we have identified a number of proteins that interact with Cyclophilin A (CypA), Cyclophilin B (CypB) and FK-Binding Protein 12 (FKBP12) by yeast two hybrid assays. Some of those novel interactions were confirmed by pull-down and co-immunoprecipitation assays. Finally, we have also carried out functional assays in some of those interactions. As a conclusion, we could suggest that cyclophilins would be involved in the nephrotoxic effects caused by cyclosporine A. Those effects are mediated by alternative pathways. Those pathways would be integrated by novel effectors such as the sodium/potassium ATPase, mitochondrial ATP synthase and also other proteins that will be studied in our lab.
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Význam V-ATPasy pro rezistenci nádorových buněk / Importance of V-ATPase for cancer cell resistenceSuchánková, Kristýna January 2021 (has links)
Chemoresistance is one of the main causes of failure of anticancer chemotherapy. Vacuolar-type ATPase (V-ATPase) is an ATP-dependent proton pump involved in the regulation of the pH in cells, cell organelles and the intracellular space. A significant acidification of the extracellular space and intracellular compartments occurs in connection with the metabolism of tumour cells (glucose metabolism, hypoxia, insufficient blood perfusion of the cancer tissue). Basic drugs are transferred into acidic organelles based on the pH gradient, where they are then protonated and accumulated. This mechanism is called lysosomal sequestration and is one of the mechanisms how tumour cells resist to applied drugs, which then do not reach their target site in cancer cell. An increased expression of V-ATPases has been described in relation to chemoresistance and the progression of tumours. This dissertation is focused on observing the membrane subunit V0d from the complex of V-ATPase and the changes in resistance to ellipticine caused by the silencing of this subunit's gene in human neuroblastoma cell lines UKF-NB-4 (sensitive) and UKF-NB-4ELLI (resistant to ellipticine). The expression of the V0d protein was first examined on mRNA level using real-time polymerase chain reaction (RT-PCR). The silencing of selected...
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Organización morfológica e histoquímica de los distintos tipos de fibras que integran el músculo flexor carporradial del perroLatorre Reviriego, Rafael 21 December 1990 (has links)
Mediante diferentes técnicas histoquímicas, se analizan los tipos de fibras de la musculatura esquelética (Mm. semitendinoso y flexor carporradial) de perros adultos y cachorros (0-65días). Tomando el músculo semitendinoso como patrón, se establecen los criterios para la correcta identificación de las distintas fibras. En base a la actividad mATPasa se reconocen 4 tipos diferentes, que denominamos: I, IIA, IIp y IIC. Con idéntica metodología y criterios de interpretación, se procede al estudio el M. flexor carporradial. Secciones transversales completas de este músculo obtenidas en criosotato a -20ºC, fueron analizadas desde el punto de vista histoquímico, procediéndose al estudio de los tipos de fibras y distribución de las mismas. Los resultados obtenidos indican que el M. flexor carporradial presenta dos cabezas, radial y cubital, cuya funcionalidad puede ser diferente. La cabeza radial se integra casi de forma exclusiva por fibras tipo I, mientras la cabeza cubital muestra una distribución en mosaico con presencia de fibras I, IIA y IIC. Las fibras IIp no fueron identificadas en ninguna de las dos cabezas. Los resultados parecen sugerir que el M. flexor carporradial del perro no solo participa en la protracción del miembro torácico (flexión del carpo), sino que desarrolla un importante papel en el aplomo del mismo. Los estudios llevados a cabo durante el periodo postnatal (0-65días), confirman la aparición gradual y progresiva de las fibras identificadas en animales adultos, aunque en distintos periodos de tiempo según el músculo y/o cabezas. Las técnicas de m-ATPasa permiten formular una nomenclatura específica para las fibras durante el desarrollo, que puede ser tomada como referencia para futuros estudios en estos periodos. / By jeans of different histochemical techniques, fibre types of skeletal muscle (Mm. semitendinosus and flexor carpi radialis) in adults and young dogs (0-65 days) were analysed. Histochemical criteria to the right identification of the different fibres were established by using semiteninosus muscle as control. According to their m-ATPase activity, we observed four different fibre types designated as I, IIA, IIp and IIC. To study the flexor carpi radialis muscle the same techniques and histochemical criteria were followed too. Cryostat whole cross serial sections were histochemically analysed, and the fibre types as well as their distribution studied. The results showed that flexor carpi radialis muscle has two heads, radial and cubital, probably with different functionality. Radial head was almost exclusively constituted by type I fibres; on the other hand, cubitl head exhibited mosaic pattern distribution, containing I, IIA and IIC fibres. None of the heads showed IIp fibre types. These findings suggest that flexor carpi radialis muscle in dog not only participates in the protraction of the forelimb (flexes carpal joint) but also takes an important part in its own aplomb. This study which was carried out throughout postnatal period (0-65 days) confirmed that occurrence of the different fibre types is gradual and progressive in time depending on muscle and / or head portion. mATPase techniques allow us the formulation of an specific nomenclature for the fibres during their development thus, it is recommended for further studies of muscle development.
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Studium poruch cytochrom c oxidasy a ATP synthasy na biochemické a molekulární úrovni / Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficienciesFornůsková, Daniela January 2011 (has links)
Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...
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Studium membránových receptorů pomocí vazby radioligandů / The study of membrane receptors by radioligands bindingRejhová, Alexandra January 2011 (has links)
Drug addiction, opiates respectively, is a social problem which seriousness is currently on the rise. One of key elements causing addiction is tolerance to increasing doses of drug causing abstinence syndrome during withdrawal and craving. Opioid receptors are members of a large group of receptors coupled with heterotrimeric G-proteins (GPCR), whose properties can be investigated using agonist- stimulated binding [35 S] GTPγS. Many extracellular signals are transferred into a cell through GPCR. Opioid receptor agonists inhibit the activity of adenylyl cyclase and are coupled with G-protein group Gi/Go. This work is devoted to the study of changes in isolated plasma membranes of rat forebrain containing opioid receptors of healthy subjects with membranes acquired from morphine addicted subjects. The rats were long-term morphine treated in increasing doses, to develop the dependency. The comparison is done firstly by binding of [3 H]ouabain to Na,K-ATPase, which proves to be a negative standard of changes, secondly by binding [35 S]GTPγS to G-proteins, thereby providing the functional activity of G-protein in stimulating the binding by the agonist of δ-opioid receptors DADLE or agonist of µ-opioid receptors DAMGO. Furthermore, it has been studied the influence of prostaglandin E1 on binding [35...
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Studium poruch cytochrom c oxidasy a ATP synthasy na biochemické a molekulární úrovni / Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficienciesFornůsková, Daniela January 2011 (has links)
Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...
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